Article pubs.acs.org/JPCC
Enhanced Enzymatic Activity of OPH in Ammonium-Functionalized Mesoporous Silica: Surface Modification and Pore Effects Kheireddine El-Boubbou,† David A. Schofield,‡ and Christopher C. Landry*,† †
Department of Chemistry, University of Vermont, 82 University Place, Burlington, Vermont 05405, United States Guild Associates Inc., 1313 Ashley River Road, Charleston, South Carolina 29407-5315, United States
‡
S Supporting Information *
ABSTRACT: Organophosphorus hydrolase (OPH) is an enzyme capable of rapidly degrading organophosphorus toxins, such as phosphorus-based nerve agents and pesticides. In these studies, OPH was immobilized within the pores of spherical mesoporous silica particles and the rate of hydrolysis of diethyl-4-nitrophenyl phosphate (paraoxon) was correlated to surface functionalization (acid, amine, or ammonium) and pore diameter. Conversion rates were measured by using a novel 31P NMR method developed by our group. After immobilization within ammonium-modified particles, OPH was more active for paraoxon degradation than free OPH, while amine- or acid-modified particles led to decreased activities. OPH immobilized within the pores of intermediate diameter (6 nm) also showed the highest activities. We also varied the amount of ammonium coverage and found that 14−20% w/w of the ammonium organosilane per particle was sufficient to observe enhancement of OPH activity.
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INTRODUCTION Porous materials have extensive internal surface areas that can be used for immobilization of active catalytic species, particularly enzymes.1 Mesoporous silica is a particularly attractive material for this application because it is prepared by a self-assembly process that allows key physical properties such as pore size, particle morphology, and particle diameter to be controlled.2,3 This allows repeated cycles of optimization to be performed, so that the structure of the porous material can be tailored for specific applications. The flexibility in creating silica-based mesoporous materials4 with a range of internal pore volumes, controllable pore diameters, and large surface area has led many researchers to study their potential as adsorbents of biomolecules for various medical and catalytic applications.5 Specifically, the pore diameters of the acid-prepared mesoporous silica (APMS)6 are normally in the range of 2 to 20 nm, which are on the same scale as enzymes and even their molecular substrates. This creates a reactive area within which solvent and substrate are trapped in intimate contact with the surface, an effect that can lead to rapid and unique reactivities. Moreover, since the enzymes interact with the surface, functionalization plays an important role in enhancing enzyme reactivities and increasing protein loading densities.1,7−10 Several recent publications have demonstrated the use of enzymes immobilized onto porous silica particles to degrade environmental toxins, such as organophosphonates.11−14 The toxicity of organophosphonates has been known for many years; compounds such as paraoxon, parathion, and malathion were widely used as insecticides in the mid-20th century (Figure 1).15 Related compounds such as sarin, soman, and “VX”, with their significantly higher human toxicity, are classified as chemical warfare agents (CWA) and were stockpiled by the United States and the Soviet Union prior to 1970.16 The biological hazard of © 2012 American Chemical Society
these compounds arises from their action as acetylcholinesterase inhibitors, leading to buildup of acetylcholine in nerve clefts, paralysis, and rapid death.17,18 Methods of decomposing organophosphonates based on the bacterial enzyme organophosphorus hydrolase (OPH) have recently been developed.19−21 Interestingly, it has also been discovered that immobilizing OPH within the pores of functionalized mesoporous silica particles increased the activity of the enzyme.11−13 We reasoned that, in addition to this effect, another potential benefit of trapping OPH within porous silica particles was that the constrained environment and electrostatic surface interactions which hinder the denaturation of the enzyme, and provide protection from external sources of contamination, allow it to be stored for an extended shelf life. In our previous work, we developed a thermally stable decontamination system consisting of OPH loaded into APMS with 6 nm pores functionalized with quaternary ammonium (APMS-amm), in which 31P NMR spectroscopy was used to monitor enzymatic conversions and measure reaction rates.14 OPH immobilized within APMS-amm retained activity after being heated to temperatures as high as 65 °C for 2−24 h whether heated in solution or dry; in contrast, free OPH showed little or no activity after identical treatments. The immobilized enzyme was also still active after repeated heating cycles. In an extreme test, OPH showed activity after being heated dry at 45 °C for 1 month. These results indicated that the ammonium functionalization and the confined environment within the pores imparted enhanced activity and stability to the enzyme. Received: March 10, 2012 Revised: July 21, 2012 Published: July 24, 2012 17501
dx.doi.org/10.1021/jp3023309 | J. Phys. Chem. C 2012, 116, 17501−17506
The Journal of Physical Chemistry C
Article
Figure 1. Structures of organophosphorus pesticides and nerve agents.
Preparation of Samples for Ammonium Experiments (APMS-amm and Ammonium Ions). The different ammonium siloxane and salt solutions (0.0116 mmol, same molar equivalence of ammonium found on APMS-6-amm, 20% silane) were incubated with 1 mL of the same stock OPH solution and gently shaken at room temperature for 4 h. The abbreviations for the ammonium controls are as follows: ammonium carbonate (AmCb), ammonium chloride (AmCl), ammonium acetate (AmAc), ammonium bromide (AmBr), tetraethyl ammonium bromide (TEAB), tetrabutyl ammonium bromide (TBAB), dodecyltrimethyl ammonium bromide (DDTMAB), didodecyldimethyl ammonium bromide (DDDAB), cetyltrimethyl ammonium bromide (CTAB), N-(trimethoxysilylpropyl)N,N,N-trimethylammonium chloride (SiMeAmCl), N-trimethoxysilylpropyl-N,N,N-tri-n-butylammonium chloride (SiBuAmCl), and tetradecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride (SiDDAmCl). Enzymatic Activity of OPH in the Presence of APMSamm and Ammonium Ions. OPH enzymatic activity was measured in 1 mM paraoxon in Tris buffer (100 mM, pH 7.4), using 31P NMR. Paraoxon (200 μL, 5 mM in MeOH) was added to OPH-loaded APMS (3 mg in 800 μL Tris buffer, 3.5 μg OPH/ mg silica, 10.5 μg OPHtotal) or ammonium-incubated OPH solutions (10.5 μg OPHtotal in Tris buffer), the mixture was gently shaken at 500 min−1 for 1 min, and the percent 31P NMR conversions were recorded. The enzymatic activity was monitored by recording the integration of the phosphorus peak shift of the formed diethyl hydrogen phosphate product at −2.8 ppm with respect to paraoxon substrate at −10.2 ppm (percent conversion = product peak integral/(substrate + product peak integral) × 100). Enzymatic Activity of Surface-Modified APMS Samples. Aqueous OPH solution (4 mL, 0.133 mg/mL, 89% pure, 41.5 units/mg paraoxon activity) was incubated with functionalized APMS (150 mg) having different pore diameters but with the same weight percent of organosilane modification (∼17 wt % by thermogravimetric analysis (TGA) and gently shaken at 500 min−1 on an Eppendorf Thermomixer at room temperature for 4 h. OPH loading in APMS was found to be 3.55 μg OPH/mg APMS (i.e., all of the OPH in the original solution was loaded). The errors in all enzyme concentration calculations were 4 nm). OPH loaded into acid-modified materials showed the least activity (Figure 4d); moreover, calcined or extracted APMS (i.e., no surface modification) also did not induce an increase in activity. These results indicate that a combination of confinement effects and surface interactions is responsible for the increase in OPH activity. OPH is a roughly globular, dimeric enzyme with dimensions of 6.1 nm × 8.6 nm × 5.1 nm and thus cannot fit easily within 4 nm pores without distortion, leading to decreased activity.26 On the other hand, larger pores (8 nm) allow OPH to be adsorbed but do not provide the same confinement as 6 nm pores. For this reaction, a close match between OPH diameter and pore diameter appears to be the best for increasing enzymatic activity.
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CONCLUSIONS In conclusion, a convenient and effective system for decontaminating organophosphorus toxins has been developed. OPH immobilized within APMS particles that were covalently modified with an ammonium organosilane was significantly more active toward the hydrolysis of paraoxon than free OPH. The amount of organosilane modification was important, with 14 wt % ammonium or higher being sufficient to significantly increase OPH activity. Although the same amount of free ammonium organosilane could increase OPH activity somewhat, the localization of the ions on the surface produced the largest effect. Of the three types of surface modifications tested, only the ammonium organosilane increased OPH activity after immobilization; amine and acid functionalities did not. We also found that a pore diameter of 6 nm was optimum for increasing OPH activity. In general, immobilized OPH can be tested against a range of organophosphorus compounds, potentially leading to the development of new protection and decontamination systems.
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ASSOCIATED CONTENT
S Supporting Information *
Synthesis of APMS and subsequent functionalization and SEM images. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: +1 802 656 8705. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by the Defense Threat Reduction Agency through grant no. HDTRA1-080-1-0035. We thank Bruce Deker for the help with 31P NMR, Mohammad El-Dakdouki for his assistance in ζ potential measurements, and Michele Von Turkovich for her help with TEM.
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REFERENCES
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dx.doi.org/10.1021/jp3023309 | J. Phys. Chem. C 2012, 116, 17501−17506
